EGFR Blood Monitoring

ABSTRACT

Improved methods of assessing status of a solid-tumor cancer in a subject involving detection of tumor-associated mutations in the subject&#39;s blood.

PRIOR RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/774,946, filed Mar. 8, 2013, which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Germline and somatic mutations affecting various cell proliferationpathways are known to affect the development of cancer in patients. Forexample, the acquisition of somatic mutations that confer growthadvantage on the cells possessing such mutations is considered animportant factor in the emergence and progression of cancerous tumors.As a number of such mutations was identified, the therapies weredeveloped that target the proteins encoded by the mutated genes, as wellas the therapies targeting the signaling pathways in which these mutatedgenes are involved. As these targeted therapies were implemented intoclinical practice, it was discovered that mutations conferring theresistance to the targeted therapies develop and accumulate in thepatients' cancerous tumors, over time rendering the therapy ineffectiveand making it necessary to change the course of treatment.

One example of a solid tumor cancer in which somatic tumor mutations areknown to play an important role is lung cancer, which is a leading causeof cancer-related mortality in many countries, including the UnitedStates. Approximately 75% of lung cancer cases belong to non-small celllung cancer (NSCLC), which has an overall 5-year survival rate ofapproximately 12%. Standard surgical treatment, as well as chemotherapyand radiotherapies are available in the field of NSCLC. However, themajority of the NSCLC cases are initially diagnosed at the inoperablelate stage, and relapse is common following surgery, chemotherapy,radiotherapy and other treatments. Accordingly, treatment and diagnosisof NSCLC is a challenging medical problem. One attempt at addressing theproblem was the development of the targeted drug therapies thatinterfere with the signaling of epidermal growth factor receptor (EGFR).EGFR, which is a member of the growth factor receptor family of tyrosinekinases, is involved in signaling pathways related to cell division andis implicated in NSCLC development and progression.

Small molecule drugs erlotinib and gefitinib, which inhibit tyrosinekinase activity of EGFR, were evaluated and approved for treatment oflate stage NSCLC. It was discovered, however, that these drugs were noteffective in the majority of NSCLC patients, but are most effective in asubset of patients whose tumors contain somatic EGFR mutations that leadto an increase in the tyrosine kinase activity of EGFR. This type ofmutations is often termed “activating.” Somatic EGFR mutations that leadto resistance to tyrosine kinase inhibitor therapy in NSCLC patientswere also discovered. This type of mutations is often termed“resistance.” Resistance mutations in EGFR tend to arise in NSCLCpatients during the course of tyrosine kinase inhibitor treatment. Inthe cases of NSCLC that cannot be effectively treated by tyrosine kinaseinhibitor therapy, such as erlotinib and gefitinib, chemotherapy remainsthe most effective treatment to prolong survival. To improve the chancesof selecting an effective treatment for NSCLC patients, it is thereforeimportant to determine whether the patients' NSCLC tumors containsomatic EGFR mutations that confer sensitivity or resistance to tyrosinekinase inhibitor therapy.

The above example of the role of EGFR somatic mutations in thedevelopment of NSCLC illustrates how detecting the presence or emergenceof certain mutations in the cancerous tumors is generally important forchoosing an effective cancer treatment. For example, detecting thepresence or emergence of somatic mutations leading to targeted drugtherapy resistance in cancer patients is essential for monitoring thetherapy and assessing disease progression. Therefore, it is generallybeneficial to develop convenient and reliable methods of testing forsomatic mutations in the tumors of the cancer patients in order toimprove cancer assessment, including, but not limited to, diagnostics,monitoring, and treatment selection in such patients.

One way of detecting such mutations is testing tumor samples obtainedthrough biopsy or surgery for the presence of mutant sequencesassociated with cancer development. However, tumor tissue samples maynot be immediately available for testing. To avoid delay in detection ofthe cancer-associated mutations and selection of appropriate treatmentas well as to spare the patients from invasive procedures, it isbeneficial to develop more expedient and less invasive methods fordetecting mutations in the tumors of the cancer patients.

It is known that tumor cells circulate in the blood of patients withsolid tumor cancers, thus making it possible to detect somatic tumormutations in the blood samples of cancer patients, including detectionof EGFR mutations in NSCLC patients. However, it is difficult toreliably adapt such detection for meaningful clinical and diagnostic usedue to the small amount of circulating mutated sequences, background ofnon-mutated sequences and high levels of genomic DNA (gDNA) circulatingin the blood, the gDNA originating from broken white blood cells (WBC).Detection of mutated nucleic acid sequences originating from tumor cellsin blood samples, such as detection of EGFR mutations in NSCLC patients,suffers from inaccuracies, such as relatively high false negativedetection rates, and often requires cumbersome analytical techniquesthat may involve, for example, isolation of blood-circulating tumorcells prior to detection, or enrichment of the content of mutated DNAsequences in the sample prior to detection. Quantitative detection canbe even more difficult, due to high background DNA levels, among otherthings. It is therefore important to develop improved methods ofdetection of mutated tumor nucleic acid sequences in the blood of cancerpatients, such as detection of mutated EGFR nucleic acid sequences inthe blood of NSCLC patients, to make such detection methods useful forassessment of cancer in clinical and diagnostic practice.

BRIEF SUMMARY OF THE INVENTION

Described herein are improved methods of assessing status of a subjectwith a solid tumor cancer, comprising detecting presence or absence ofone or more tumor nucleic acid mutations in a blood of the subject withthe solid tumor cancer; and, assessing the status of the subject withthe solid tumor cancer based on the detected presence or absence of theone or more tumor nucleic acid mutations. The improved methods mayinvolve detection of the one or more tumor nucleic acid mutations byperforming a quantitative real-time polymerase chain reaction (PCR) on ablood sample or on a total genomic DNA isolated from a blood sample,where the blood sample is obtained from a subject with a solid tumorcancer. Also described herein are improved methods of detecting presenceor absence of an tumor mutation in a blood sample obtained from asubject with a solid tumor cancer, comprising performing a quantitativereal-time polymerase chain reaction (PCR) on the blood sample usingprimers specific for a mutated nucleic sequence to generate a PCR cyclethreshold. In some embodiments of the improved methods described herein,a metastatic status of the subjects' with a solid tumor cancer is takeninto account in order to improve sensitivity of the detection of themutated tumor nucleic acid sequences in the blood samples obtained fromthe subjects. In some other embodiments, detection of the presence orthe absence of the one or more tumor nucleic acid mutations in the bloodsamples obtained from the subjects with the solid tumor cancers involvesdetermining the amount of the mutated sequences circulating in the bloodand monitoring the status of the subject's cancer based on the detectedamount.

Described herein are methods of assessing status of a subject withdistant metastasis NSCLC, comprising: detecting presence or absence ofone or more mutated EGFR nucleic acid sequence in blood from the subjectwith distant metastasis stage NSCLC; and assessing the status of thesubject with distant metastasis stage NSCLC based on the detectedpresence or absence of the one or more mutated EGFR nucleic acidsequence. Also described herein are methods of assessing status of asubject with NSCLC, comprising: detecting presence or absence of one ormore mutated EGFR nucleic acid sequence in a blood of the subject; andassessing the status of the subject based on the detected presence orabsence of the one or more mutated EGFR sequence. Also described hereinare method of identifying a candidate NSCLC patient for a targeted drugtherapy, comprising: detecting presence or absence of one or moremutated EGFR sequence in blood from the patient; assessing metastaticstatus of the NSCLC patient as M1a or M1b; and identifying the patientas a candidate for the targeted drug therapy based on at least thedetected presence of the one or more mutated EGFR sequence in the bloodof the patient, and the metastatic status of NSCLC in the patient. Alsodisclosed herein are methods of assessing status of a subject with asolid tumor cancer, comprising: detecting presence or absence of one ormore tumor-associated mutated nucleic acid sequence in blood from thesubject with the solid tumor cancer; and assessing the status of thesubject with distant metastasis solid tumor cancer based on the detectedpresence or absence of the one or more mutated tumor-associated nucleicacid sequence. Furthermore, disclosed herein are methods of detectingpresence or absence of a tumor-associated mutation in a blood sample,the methods comprising: performing a quantitative real-time polymerasechain reaction (PCR) on the blood sample using primers specific for amutated nucleic sequence to generate a PCR cycle threshold; andcomparing the cycle threshold to a control value, wherein the controlvalue takes into account the concentration of genomic DNA in the sample,and wherein if the cycle threshold is below the control value thetumor-associated mutation is present in the sample and if the cyclethreshold is above the control value the tumor-associated mutation isabsent from the sample. Methods of treating patients or subjects withsolid tumor cancers, such as NSCLC, are also envisioned and includedwithin the scope of the methods described herein. Variations andcombinations of the above methods and their various steps and substepsare also envisioned and included within the scope of the describedmethods.

DEFINITIONS

The term “subject” as used herein typically refers to a person (human)having a solid tumor cancer, such as NSCLC. It is to be understood, thata subject having a solid tumor cancer can be a patient with a knowncancer, meaning the cancer that was detected prior to the performance ofthe embodiments of the methods of the present invention. A cancerpatient can be a relapse cancer patient. For example, a subject havingNSCLC can be a patient in whom NSCLC was detected prior to theperformance of the embodiments of the methods of the present invention.The NSCLC patient can be a relapse patient.

The terms “recurrent,” “recurrence,” “relapsed,” “relapse” and relatedterms are used to refer to cancer that returns after treatment, and tothe patients that experience the return of the cancer.

The term “solid tumor cancer” is used herein to denote the cancers thatare characterized by the formation of cancerous tumors, or cohesivemasses of abnormally proliferating cells, in tissues and organs. It isto be understood that some tumors formed by the solid tumor cancers canbe cysts, meaning fluid-filled sacks of tissue. The term “solid tumorcancer” is used herein to distinguish tumor-forming cancers from theso-called blood cancers or hematological malignancies that are formedfrom hematopoietic (blood-forming) cells and affect blood, bone marrow,and lymph nodes. Examples of solid tumor cancers are carcinomas, orcancers derived from epithelial cells, sarcomas, or cancers arising fromconnective tissue, germ cell tumors, such as seminomas anddysgerminomas, blastomas, or cancers that derive from precursor cells orembryonic tissue. Some non-limiting examples of solid tumor cancers arelung cancer, breast cancer, colorectal cancer, prostate cancer, thyroidcancer, brain cancer, such as glioblastoma, and bladder cancer. Examplesof hematological malignancies are lymphomas, leukemias, myelomas,myelodysplastic syndromes and myeloproliferative diseases.

The term “therapy” is used herein synonymously with the term“treatment.” The term “cancer therapy” as used herein encompassesvarious types of cancer therapy or treatment, including surgery,radiotherapy, chemotherapy, and targeted drug therapy.

“Targeted therapy” or “targeted drug therapy” refer to drug therapy thatinterferes with the growth of cancer cells by interfering with specificmolecules needed for carcinogenesis and tumor growth, rather than bysimply interfering with all rapidly dividing cells, as chemotherapydoes. An example of targeted drug therapy is tyrosine kinase inhibitortherapy, which uses reversible tyrosine kinase inhibitors to inhibit theactivity of tyrosine kinases promoting cell proliferation in certaintypes of cancers. For example, erlotinib or gefitinib target tyrosinekinase activity of EGFR and are used as a targeted therapy for non-smallcell lung cancer.

The term “targeted drug therapy,” as used herein, is not limited to theabove therapies, but can encompass any drug therapy interfering with aspecific target, such as therapies that interfere with EGFR signaling.Targeted drug therapies include, but are not limited to, reversibletyrosine kinase inhibitor therapy, irreversible tyrosine kinaseinhibitor therapy, antibody therapy, or any form of small molecule,large molecule or nucleic-acid based therapy, such as gene therapy orsmall interfering RNA therapy.

The term “tumor-associated mutation” is used herein to denote mutationsin nucleic acid sequences that affect development of a solid tumorcancer in a subject. For example, a tumor-associated mutation canactivate cellular proliferation, thus leading to emergence of amalignant tumor or escalation of tumor growth. A tumor-associatedmutation can confer properties on a tumor that facilitate its spreadthroughout the subject's body, known as metastasis. A tumor-associatedmutation can also be associated with susceptibility or resistance of acancer to cancer therapies. The term “tumor-associated” can be used inreference to nucleic acids or nucleic acid sequences comprising one ormore tumor-associated mutations, such as in an expression“tumor-associated mutated nucleic acid sequence.”

The terms “assess,” “assessment,” “assessing” and the related terms areused herein in reference to cancer, status of cancer or status of asubject with cancer, and in some other contexts. These terms can denotebut are not limited to inferring the presence or the absence ofcancer-associated mutations in cancerous tumors based on the detectedpresence or absence of mutated nucleic acid sequences in the subject'sblood. The terms “assess,” “assessment,” “assessing” and the relatedterms may also encompass, depending on the context, recommending orperforming any additional diagnostic procedures related to evaluatingthe presence or absence of cancer-associated mutations in the subject'stumors, evaluating potential effectiveness of the treatments for thesubject's cancer as well as recommending or performing such treatments,monitoring the subject's cancer, or any other steps or processes relatedto treatment or diagnosis of a cancer.

The expressions “detect in blood,” “detection in blood,” “detecting inblood,” and the related expressions, as used herein, refer to the act orthe result of finding or discovering nucleic acid sequences in a sampleof the liquid fraction of blood, such as plasma or serum.

The term “local metastasis” to a process or a result of a process, inwhich cancer cells originating from a cancerous tumor penetrate andinfiltrate surrounding normal tissues in the local area, typically inthe same or adjacent organ or organs, forming new tumor. For example,“local metastasis” metastatic stage of NSCLC means that metastasis ispresent, but no metastasis is detected in extrathoracic organs. Inreference to NSCLC, the term “local metastasis” encompasses themetastatic stage “M1a.”

The term “distant metastasis” refers to a process or a result of aprocess, in which cancer spreads to tissues and organs that are distantfrom the primary tumor site. For example, the term “distant metastasis”used in the context of NSCLC means that metastasis is present and isdetected in extrathoracic organs. In reference to NSCLC, the term“distant metastasis” encompasses the metastatic stage “M1b.”

The terms “detect,” “detecting,” “detection,” “and similar terms areused in this document to broadly to refer to a process or discovering ordetermining the presence or an absence, as well as a degree, quantity,or level, or probability of occurrence of something. The termsnecessarily involve a physical transformation of matter such as nucleicacid amplification. For example, the term “detecting” when used inreference to EGFR mutation, can denote discovery or determination of thepresence, absence, level or quantity, as well as a probability orlikelihood of the presence or absence of the EGFR mutation. It is to beunderstood that the expressions “detecting presence or absence,”“detection of presence or absence” and related expression, when used inreference to tumor-associated mutations, include qualitative andquantitative detection. Quantitative detection includes thedetermination of level, quantity or amounts of mutated nucleic acidsequences in the sample, on which the detection process is performed.

The term “mutation” or “mutated sequence,” when used in reference tonucleotide or amino acid sequence can be used interchangeably with theterms “variant,” “allelic variant,” “variance,” or “polymorphism.” Forexample, the phrases “detecting a mutation,” “detecting a mutatedsequence” “detecting polymorphism” or “detecting sequence variance” canbe used interchangeably when discussing the methods of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of some known EGFR mutations found inthe tyrosine kinase domain of EGFR, adapted from Sharma et al., Nat.Rev. Cancer, 7:169 (2007).

FIG. 2 is a plot illustrating experimental data on real-time PCRcross-point (Cp) values obtained with COBAS® EGFR Mutation Test kitusing reaction mixtures MMX1, MMX2 and MMX 3 in the presence ofdifferent levels of genomic DNA. The X-axis represents genomic DNA levelC_(p) per reaction) and the Y-axis represents cycle number correspondingto the C, achieved in a reaction.

FIG. 3 is a plot schematically showing an exemplary calibration curvefor quantification of a target nucleic acid.

FIG. 4 is a schematic representation of NSCLC treatment timeline andsample collection.

FIG. 5 is a plot illustrating detection of EGFR mutations in the plasmasamples of two exemplary NSCLC patients. Week 0 on the X-axiscorresponds to time point CP₀ before the start of erlotinib treatment inFIG. 3.

FIG. 6 is a schematic representation of the decision-making process fortreatment and diagnosis of patients presenting with NSCLC patients basedon blood testing for EGFR activating mutations.

FIG. 7 is a schematic representation of the decision-making process fortreatment and diagnosis of relapsing NSCLC patients based on bloodtesting for EGFR activating mutations.

FIG. 8 is a schematic representation of the decision-making process fortreatment and diagnosis of NSCLC patients.

DETAILED DESCRIPTION OF THE INVENTION

The subject matter of embodiments of the present invention is describedhere with specificity to meet statutory requirements, but thisdescription is not necessarily intended to limit the scope of theclaims. The claimed subject matter may be embodied in other ways, mayinclude different elements or steps, and may be used in conjunction withother existing or future technologies. This description should not beinterpreted as implying any particular order or arrangement among orbetween various steps or elements except when the order of individualsteps or arrangement of elements is explicitly described.

The inventors have discovered that detection of tumor-associated mutatednucleic acid sequences circulating in the blood of a subject with asolid tumor cancer can be performed quickly and accurately by performingreal-time quantitative PCR on the blood sample or on the genomic DNAisolated from a blood sample obtained from the subject with the solidtumor cancer. By improving the methods of processing and analyzingquantitative PCR data, the inventors achieved unexpectedly improvedvalidity of the measurements of tumor-associated mutated nucleic acidsequences circulating in the blood of the subjects with solid tumorcancers. The inventors discovered that a status of a solid tumor cancerin a subject can be advantageously assessed by measuring the type andamount of tumor-associated mutated nucleic acid sequences circulating inthe subject's blood. The inventors have also discovered that detectionof tumor-associated mutations in subjects with solid tumor cancers basedon the detection of mutated nucleic acid sequences circulating in thesubjects' blood can be significantly improved if the metastasis statusof the cancer in such subjects is taken into account.

Detection of Tumor-Associated Mutations in Blood that Takes into AccountMetastatic Status of a Subject

In one example, the inventors have discovered that detection of EGFRmutations in NSCLC subjects based on the detection of mutated EGFRsequences circulating in the subjects' blood can be significantlyimproved if the metastasis status of the NSCLC subjects is taken intoaccount. In particular, the inventors have discovered that in a subsetof NSCLC subjects, those subjects having distant metastasis NSCLC, thepresence or absence of EGFR mutations detected by amplification ofnucleic acid present in blood accurately predicts the presence orabsence of EGFR mutations in the subjects' NSCLC tumors. In view of thediscovery that blood assays are reliable for subjects with distantmetastases, a negative result, i.e., a finding of no EGFR mutations in ablood sample, is sufficient to determine that the subject does not carrythe EGFR mutation and therefore does not require an invasive biopsy toconfirm the negative results. In contrast, in NSCLC subjects withoutdistant metastasis NSCLC, while the presence of detectable EGFRmutations in blood serves as an accurate predictor of the presence ofEGFR mutations in the subjects' NSCLC tumors, the absence of detectableEGFR mutations in blood cannot serve as an accurate predictor of theabsence of EGFR mutations in the subjects' NSCLC tumors.

The above discovery can be generally applied to the detection oftumor-associated mutations in the blood of the subjects with solid tumorcancers. Detection of an absence of a tumor-associated mutation in ablood sample obtained from a subject with distant metastasis solid tumorcancer is sufficient to determine the subject does not carry themutation and therefore does not require any additional procedures, suchas an invasive biopsy, to confirm the negative results. In contrast, ifa subject has a solid tumor cancer without distant metastasis, detectionof a presence of a tumor-associated mutation in blood serves as anaccurate predictor of the presence of the mutations in the subjects'tumors, while detection of the absence of a detectable mutation in bloodcannot serve as an accurate predictor of the absence of the mutation inthe subjects' tumors. Accordingly, described herein are methods thatdetect the presence or absence of tumor-associated mutations in theblood of a subject with a solid-tumor cancer, in order to assess thesubject's status. Some embodiments of the above methods are the methodsthat detect the presence or absence of mutations in epidermal growthfactor receptor (EGFR) in the blood of a subject with non-small celllung cancer (NSCLC), in order to assess the subject's status.

Tumor-associated mutations can affect the effectiveness of cancertreatments. For example, tumor EGFR mutations influence theeffectiveness of certain NSCLC treatments, such as therapies targetingEGFR, for example, tyrosine kinase inhibitor therapies, including, butnot limited to, erlotinib and gefitinib. By using the methods describedherein, the mutation status of the cancerous tumors in the subject canbe accurately assessed and applied to the decision-making process onselection and administration of appropriate therapy, if any exists, oradditional diagnostic procedures.

Before the discoveries described herein, high false negative error ratelimited application of blood-based detection of tumor-associatedmutations in a clinical and diagnostic context, since it necessitatedadditional testing of tumor tissue of the patients foundmutation-negative based on the blood samples. Some embodiments of themethods described herein address the above problem by discriminatingsolid-tumor subjects based on their metastasis status. In particular,the methods described herein incorporate and apply the discovery thatthe high false negative rate observed in the previously describedblood-based diagnostic procedures is not observed among the subjectswith metastatic NSCLC distant metastasis (e.g., M1b metastasis status).Blood detection of EGFR mutations in M1b metastasis status NSCLCsubjects can therefore be used as a reliable diagnostic procedure forNSCLC monitoring and in determining further course of diagnosis andtreatment of NSCLC.

The embodiments of methods described herein are not limited to diagnosisand treatment of NSCLC subjects, but are generally applicable todiagnosis and treatment of the subjects with various solid-tumorcancers. Furthermore, embodiments of the methods described herein arenot limited to the subjects with distant metastasis solid-tumor cancer.According to some embodiments of the methods described herein, thestatus of the solid-tumor in the subject without distant metastasis canalso be assessed. The assessment involves inferring whether or not thesubjects' tumor tissue contains mutations detected in the blood usingthe following criteria. The presence of the mutated sequence in theblood of the subject with a solid tumor cancer but without distantmetastasis indicates a high likelihood that the subject's tumor tissuecontains the mutations detected in the blood. Therefore, if mutantsequences are detected in a blood of a subject without distantmetastasis (such as in a subject with no metastasis or only localmetastasis), further diagnostic and treatment decisions can be madebased on the high likelihood of the presence of the mutations in thesubject's tumor. However, the absence of the sequence in the blood ofthe subject with solid tumor cancer but without distant metastasis doesnot reliably indicate that the subject's tumor tissue does not containthe mutations detected in the blood. If mutant sequences are notdetected in a blood of such a subject, then additional diagnosticprocedures are warranted to ascertain the presence of mutations in thesubject's tumors.

For example, when the above embodiments of the methods of assessing astatus of a subject with a solid-tumor cancer are applied to NSCLCsubjects, the following decision-making process can be performed. Thepresence of the mutated EGFR sequence in the blood of the subject withNSCLC but without distant metastasis indicates a high likelihood thatthe subject's NSCLC tumor tissue contains the EGFR mutations detected inthe blood. Therefore, if EGFR mutant sequences are detected in a bloodof a subject without metastatic NSCLC of stage M1b, further diagnosticand treatment decisions can be made based on the high likelihood of thepresence of the EGFR mutations in the subject's tumor. However, theabsence of the sequence in the blood of the NSCLC subject withoutdistant metastasis does not reliably indicate that the subject's NSCLCtumor tissue does not contain the EGFR mutations detected in the blood.If EGFR mutant sequences are not detected in a blood of such a subject,then additional diagnostic procedures are warranted to ascertain thepresence of mutations in the subject's NSCLC tumors.

Methods of Monitoring a Solid Tumor Cancer in a Subject by DetectingTumor-Associated Mutated Sequences in the Subject's Blood

The methods of assessing status of a subject with a solid-tumor cancerdescribed herein include diagnostic methods that use detection oftumor-associated mutations in a blood of a subject to monitor status andprogression of the solid-tumor cancer in the subject. Included withinthe embodiments of the above methods are the diagnostic methods that usedetection of EGFR mutations in a blood of a NSCLC subject to monitorNSCLC status and progression in the subject.

The determination according to the above methods can be an in vitrodetermination performed on a blood or plasma sample extracted from thesubject. The determination can be useful for monitoring cancer therapyeffects and making decisions on cancer therapy selection. For example,the methods described herein can be used before, during and/or aftertumor-removal surgery on a subject, to monitor the surgery'seffectiveness. The methods can also be used before, during, or after anycancer therapy. For example, the methods can be used prior to a cancertherapy to determine the likelihood of the effectiveness of the therapyin a particular subject, or identifying a subject as a suitablecandidate for a cancer therapy. The methods can be used during or aftercancer therapy to determine the therapy's effectiveness as well as tomonitor the emergence of resistance to cancer therapy. The methods canalso be used during cancer remission to monitor cancer recurrence andprogression.

In some embodiments, the methods employ qualitative detection oftumor-associated mutations to determine the presence or absence, or thenature of tumor-associated mutations in the blood of the subjects. Insome embodiments, the methods employ quantitative determination oftumor-associated mutations to determine the amount of mutated sequencespresent in the subject's blood. Qualitative or quantitativedetermination, or combination thereof, can be referred to asdetermination or detection of a “mutation load,” and can be used toassess the status of a solid-tumor cancer in the subject, including theseverity of the cancer. Mutation load of tumor-associated mutation in ablood of a subject can be characterized by the number oftumor-associated mutations in the subject's blood (that is, how manydifferent mutations are detected), amount of-tumor associated mutationsdetected in the subject's blood (quantity of the mutatedtumor-associated nucleic acids circulating in the subject's blood), or acombination of the foregoing. It is to be understood that, in somecases, a mutation load of tumor-associated mutations detected in theblood of a subject with a tumor-associated cancer correlates with thecancer's severity and/or progression in a subject. Mutation load canalso correlate with the effectiveness or lack thereof of cancertherapies administered to the subject.

In one embodiment of the methods of monitoring a solid tumor cancerdescribed herein, the mutation load being detected is quantity of atleast one activating tumor-associated mutation and at least oneresistance tumor-associated mutation in a blood sample obtained from asolid cancer patient. The mutation load is being detected over time, forexample, during a course of cancer therapy or therapies. The detectedquantity of the at least one activating tumor-associated mutation servesas an indicator of cancer progression, severity, and/or a success orlack thereof of the therapy or the therapies administered to thepatient. The detected quality of the at least one resistance mutationserves an indicator of cancer progression, severity, resistance to thetherapy or therapies being administered to the patient. Thedecision-making process on the treatment of the solid-tumor cancer inthe patient is performed based on the mutation load being detected.

Unexpectedly, by applying the methods described herein, progression,severity or stage of a solid-tumor cancer in a patient, as well assusceptibility of the cancer to certain therapies, can be reliablyrecognized or determined before the emergence of clinical signs orsymptoms in the subject, or before the signs or symptoms becomedetectable by conventional detection techniques and procedures. In somecases, the status of a solid tumor cancer in a subject can be assessedone or more (meaning 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or anyinterval delineated by these integers) weeks or months before theemergence of clinical or conventionally detectable sign or symptoms ofsolid tumor cancer in the subject. Clinical decisions can therefore bemade based on the tumor-associated mutation load found in the subject'sblood. For example, a cancer therapy can be started, stopped or changedbased the subject's detected mutation load. In another example, a cancertherapy dose can be adjusted, such as increased or decreased, based onthe subject's tumor-associated mutation load. The methods of monitoringa solid tumor cancer described herein advantageously reduce or minimizethe number of complex, expensive or invasive diagnostic proceduresperformed on a solid tumor cancer patient, while at the same timeproviding diagnostic data for informed clinical decision making process.In some cases, the methods of monitoring a solid tumor cancer describedherein can replace more expensive and/or invasive diagnostic procedures,such as biopsies. The methods of monitoring a solid tumor cancerdescribed herein can lower the cost of cancer treatment and diagnostics,decrease patient discomfort, and lead to more accurate clinical decisionmaking process, which may lead to more favorable cancer treatmentoutcomes.

In one illustrative example, the mutation load of EGFR mutations in ablood of a subject with NSCLC is determined and used in a clinicaldecision-making process. Targeted EGFR therapy is indicated andadministered to the subject based on the detected presence of one ormore activating EGFR mutations in the subject's blood. The dose of thetargeted EGFR therapy is determined based on the activating EGFRmutation load. For example, higher dose of the targeted EGFR therapywith a reversible tyrosine kinase inhibitor (“reversible TKI therapy”)can be recommended based on the higher mutation load. The status of theNSCLC subject is monitored during the course of the reversible TKItherapy. Decrease or, in some cases, maintenance of the activating EGFRmutation load indicates a success of the reversible TKI therapy,indicating that it can be continued or, in some cases, stopped. Increasein EGFR mutation load indicates a decrease in the effectiveness of thereversible TKI therapy. Emergence of the resistance EGFR mutations orincrease of resistance EGFR mutation load also indicates a decrease or apotential decrease in the effectiveness of the reversible TKI therapy.When a decrease or potential decrease in the effectiveness of the TKItherapy is detected, various clinical decisions can be made, such asincreasing the dosage of the reversible TKI therapy, administering adifferent therapy, such as chemotherapy and/or radiotherapy,administering a different targeted therapy, such as an irreversible TKItherapy, or any combination of the foregoing.

Improved Detection of Tumor-Associated Mutations

In the embodiments of the methods described herein, nucleic acidsequences are detected by suitable methods, such as quantitativeamplification. Methods of quantitative amplification are disclosed in,e.g., U.S. Pat. Nos. 5,210,015; 5,804,375; 6,127,155; 6,180,349;6,033,854; and 5,972,602, as well as in, e.g., Holland et al., Proc.Natl. Acad. Sci. 88:7276-7280 (1991), Gibson et al., Genome Research6:995-1001 (1996); DeGraves, et al., Biotechniques 34(1):106-10, 112-5(2003); Deiman B, et al., Mol Biotechnol. 20(2): 163-79 (2002).Amplifications may be monitored in “real time.”

In some embodiments of the methods described herein, quantitative PCR isemployed. Quantitative PCR refers generally to a method that allows forquantification of the amounts of the target nucleic acid sequence usedat the start at the PCR reaction. Quantitative PCR techniques usevarious approaches to quantification. One example of a quantitative PCRmethod is “real time PCR,” which can be also referred to as “real timequantitative PCR.” Although some sources use the terms “real time PCR”and “quantitative PCR” synonymously, this is not the case for thepresent document. Here, the term “quantitative PCR” encompasses allPCR-based techniques that allow for quantification of the initiallypresent target nucleic acid sequences. The term “real time PCR” is usedto denote a subset of quantitative PCR techniques that allow fordetection of PCR product throughout the PCR reaction, or in real time.The principles of real-time PCR are generally described in Holland etal. (1991) and Held et al. “Real Time Quantitative PCR” Genome Research6:986-994 (1996). Generally, real-time PCR measures a signal at eachamplification cycle. Conventional real-time PCR techniques rely onfluorophores that emit a signal at the completion of everymultiplication cycle. Examples of such fluorophores are fluorescencedyes that emit fluorescence at a defined wavelength upon binding todouble-stranded DNA, such as SYBR green. An increase in double-strandedDNA during each amplification cycle thus leads to an increase influorescence intensity due to accumulation of PCR product. Anotherexample of fluorophores used in real-time PCR is sequence-specificfluorescent reporter probes. The examples of such probes are TaqMan®probes and FRET probes. TaqMan® probes contain a fluorophore and afluorescence quencher, which reduces the fluorescence emitted by thefluorophore. During the extension phase of PCR, the probe is cleaved bythe exonuclease activity of the DNA polymerase, releasing thefluorophore. The fluorophore release results in in an increase influorescence signal, which is proportionate to the amount of the PCRproduct. FRET probes employ fluorescence resonance energy transfer(FRET). Two labeled sequence-specific probes are designed to bind to thePCR product during the annealing phase of PCR, which results in anenergy transfer from a donor fluorophore to an acceptor fluorophore.This results in an increase in fluorescence during the annealing phase,which is proportional to the amount of the PCR product.

The use of sequence-specific reporter probe provides for detection of atarget sequence with high specificity, and enables quantification evenin the presence of non-specific DNA amplification. Fluorescent probescan also be used in multiplex assays—for detection of several genes inthe same reaction—based on specific probes with different-coloredlabels. For example, a multiplex assay can use several sequence-specificprobes, labeled with a variety of fluorophores, including, but notlimited to, FAM, JA270, CY5.5, and HEX, in the same PCR reactionmixture.

One example of a multiplex assay that can be suitably employed fordetection of mutated EGFR sequences according to the methods of thepresent invention is allele-specific PCR, such the assay that can beperformed with the COBAS® EGFR Mutation Test kit (Roche MolecularDiagnostics, Indianapolis, Ind.), which employs allele-specific EGFRprimers to detect mutations in nucleic acid sequences in the presence ofwild-type variants of the sequences. Allele-specific PCR is a techniquein which the variant of the nucleic acid sequence present in the PCRreaction mixture is selectively amplified and detected. Allele-specificPCR employs at least one “allele-specific primer.” The term“allele-specific” primer generally refers to a primer whose extensionoccurs in a PCR reaction only when a specific variant of a nucleic acidsequence is present in the reaction mixture. In other words,allele-specific primers are designed in such a way that theydiscriminate between variants of nucleic acids and selectively multiplynucleic acid templates that include a variant to be detected.

Some embodiments of the methods described herein employ improveddetection methods of tumor-associated mutations in blood samplesobtained from the a subject with a solid tumor cancer. In one example,the step of detecting one or more EGFR mutations in a blood of a subjectwith NSCLC comprises detection of one or more mutated NSCLC nucleic acidsequences in a sample obtained from the subject. The detection maycomprise contacting the sample or nucleic acids isolated from thesample, such as total genomic DNA, with one more allele-specific primersand other components of a PCR, such as enzymes and nucleotides,incubating the resulting reaction mixture under the conditions allowingfor selective amplification of the mutated nucleic acid sequences, anddetecting the presence of the amplified product. Allele-specific PCR canbe combined with real-time quantitative PCR in the embodiments of themethods described herein to improve the detection of the of the mutatedtumor-associated nucleic acid sequences.

Conventional methods of detecting tumor-associated mutations in bloodsamples typically employ additional steps for increasing the content ofthe mutated sequences in the sample prior to performing PCRamplification of the mutant sequences. For example, in one conventionalmethod, isolation of tumor cells from the subject's blood sample priorto PCR amplification is performed to improve the sensitivity ofdetection of tumor-associated mutations. In another conventional method,non-mutated DNA sequences corresponding to the mutated tumor-associatedsequences are subjected to nuclease digestion prior to PCR amplificationin order to minimize the background of the non-mutated sequences.Disclosed herein are improved detection methods, which employquantitative PCR, and can detect tumor-associated mutations in bloodsamples obtained from the subjects with solid-tumor cancers, and,advantageously, do not require additional steps for isolating tumorcells, tumor DNA, or increasing the content of the mutated sequences inthe sample prior to performing real-time quantitative PCR.

As discussed above, real-time PCR relies on detection of a measurableparameter, such as fluorescence, during the course of the PCR reaction.The amount of the measurable parameter is proportional to the amount ofthe PCR product, which allows observe the increase of the PCR product“in real time.” Some real-time PCR methods allow for quantification ofthe input DNA template based on the observable progress of the PCRreaction. The analysis and processing of the data involved is discussedbelow. A “growth curve” or “amplification curve” in the context of anucleic acid amplification assay is a graph of a function, where anindependent variable is the number of amplification cycles and adependent variable is an amplification-dependent measurable parametermeasured at each cycle of amplification, such as fluorescence emitted bya fluorophore. Typically, the amplification-dependent measurableparameter is the amount of fluorescence emitted by the probe uponhybridization, or upon the hydrolysis of the probe by the nucleaseactivity of the nucleic acid polymerase, see Holland et al. (1991) Proc.Natl. Acad. Sci. 88:7276-7280 and U.S. Pat. No. 5,210,015. In a typicalpolymerase chain reaction, a growth curve comprises a segment ofexponential growth followed by a plateau, resulting in asigmoidal-shaped amplification plot when using a linear scale. A growthcurve is characterized by a “cross point” value or “C_(p)” value, whichcan be also termed “threshold value” (or C_(t) value) which is a numberof cycles where a predetermined magnitude of the measurable parameter isachieved. A lower C_(p) value represents more rapid completion ofamplification, while the higher C_(p) value represents slower completionof amplification. Where efficiency of amplification is similar, thelower C_(p) value is reflective of a higher starting amount of thetarget nucleic acid, while the higher C_(p) value is reflective of alower starting amount of the target nucleic acid. Where a controlnucleic acid of known concentration is used to generate a “standardcurve,” or a set of “control” C_(p) values at various knownconcentrations of a control nucleic acid, it becomes possible todetermine the absolute amount of the target nucleic acid in the sampleby comparing C_(p) values of the target and control nucleic acids.

The accuracy of the detection by real-time quantitative PCR thereforedepends on correct selection of a number of parameters. One parameterthat needs to be correctly determined is the range in which C_(p) valuesbear linear correlation with the starting amount of the nucleic acid,expressed in log copy number. This range can be termed “valid range” or“assay linearity range” of the real-time PCR assay.

The inventors have found that a blood sample containing genomic DNA notgenerally known to contain a tumor-associated mutation may neverthelessgenerate an amplification signal at some genomic DNA concentrations. Insome embodiments, this background level of signal is therefore a cutoffbelow which a signal must fall to be valid, i.e., to be considereddifferent from the background. As noted above, the level of backgroundamplification changes with concentration of genomic DNA. Accordingly, insome embodiments, determination of the presence or absence of atumor-associated mutation comprises comparison of a threshold value to acontrol value, wherein the control value is dependent, and varies basedupon the concentration of genomic DNA in the sample. Thus, if the cyclethreshold for the sample is below the control value then the sample isconsidered to contain the tumor-associated mutation and if the cyclethreshold of the sample is equal to or higher than the control value,the result does not indicate the presence of the tumor-associatedmutation, and can be referred to as “negative result”). In someembodiments, such as the testing of NSCLC pM1b metastatic stage patientsfor an EGFR mutation, such a negative result is indicative, with highlikelihood, of the absence of an EGFR mutation in the patients' tumors.In some other embodiments, such as testing of NSCLC patients of ametastatic stage other than pM1b (such as M0 or pM1a), for an EGFRmutation, such a negative result may not be indicative of the absence ofan EGFR mutation in the patients' tumors, and re-testing of thepatients' tumor tissue should be considered.

In some embodiments, the control value is the highest C_(p) value orrange at which non-specific amplification in the absence of the targetDNA occurs, and can be referred to as a “breakthrough” value. In someembodiments, the control value is in fact a range of values, withinwhich a positive value from a sample must fall in order to beconsidered. Said another way, the range represents possible signallevels outside the typical range of background signal. In someembodiments, the control range is between the above-describedbreakthrough value and the cycle threshold value of a positive control.In some embodiments, the control value is based on amplification of aninternal control, for example another region of the mutated locus thatis not mutated frequently.

The improved real-time quantitative PCR methods described hereinestablish the valid cycle-threshold (C_(t)) range by generating standardcurves for control DNA at various levels of genomic DNA in the real-timePCR reaction mixture and selecting the valid cycle-threshold range basedon range in which assay linearity is observed. A control or cut-offvalue for the quantitative real-time PCR reaction is determinedaccording to some other embodiments of the improved methods describedherein, below which the non-specific amplification in the absence of thetarget DNA is not likely to interfere with the quantitative detection ofthe target DNA present in the reaction mixture. In some otherembodiments, the improved methods described herein employ a calibrationcurve for quantification of a target DNA present in the reaction mixturewhich takes into account various amounts of genomic DNA present in thesample. Various combinations of improvements of real-time PCR assaysdiscussed above can be incorporated into the improved methods ofdetection of tumor-associated mutations in blood samples, or anothertarget locus in genomic DNA, thus leading to unexpectedly increasedaccuracy of such detection.

The calculations and comparisons (e.g., of a sample signal to a controlvalue or range) for the methods described herein can involvecomputer-based calculations and tools. Tools can be advantageouslyprovided in the form of computer programs that are executable by ageneral purpose computer system (referred to herein as a “hostcomputer”) of conventional design. The host computer may be configuredwith many different hardware components and can be made in manydimensions and styles (e.g., desktop PC, laptop, tablet PC, handheldcomputer, server, workstation, mainframe). Standard components, such asmonitors, keyboards, disk drives, CD and/or DVD drives, and the like,may be included. Where the host computer is attached to a network, theconnections may be provided via any suitable transport media (e.g.,wired, optical, and/or wireless media) and any suitable communicationprotocol (e.g., TCP/IP); the host computer may include suitablenetworking hardware (e.g., modem, Ethernet card, WiFi card). The hostcomputer may implement any of a variety of operating systems, includingUNIX, Linux, Microsoft Windows, MacOS, or any other operating system.

Computer code for implementing aspects of the present invention may bewritten in a variety of languages, including PERL, C, C++, Java,JavaScript, VBScript, AWK, or any other scripting or programminglanguage that can be executed on the host computer or that can becompiled to execute on the host computer. Code may also be written ordistributed in low level languages such as assembler languages ormachine languages.

The host computer system advantageously provides an interface via whichthe user controls operation of the tools. In the examples describedherein, software tools are implemented as scripts (e.g., using PERL),execution of which can be initiated by a user from a standard commandline interface of an operating system such as Linux or UNIX. Thoseskilled in the art will appreciate that commands can be adapted to theoperating system as appropriate. In other embodiments, a graphical userinterface may be provided, allowing the user to control operations usinga pointing device. Thus, the present invention is not limited to anyparticular user interface.

Scripts or programs incorporating various features of the presentinvention may be encoded on various computer readable media for storageand/or transmission. Examples of suitable media include magnetic disk ortape, optical storage media such as compact disk (CD) or DVD (digitalversatile disk), flash memory, and carrier signals adapted fortransmission via wired, optical, and/or wireless networks conforming toa variety of protocols, including the Internet.

General Considerations Applicable to the Embodiments of the PresentInvention

A subject having a solid-tumor cancer, such as NSCLC, can have thesolid-tumor cancer that was not diagnosed prior to the performance ofthe methods according to the embodiments of the present invention. Forexample, a subject can be tested for the presence of a tumor-associatedmutation, such as EGFR mutant sequences, in blood before or duringcompletion of other diagnostic procedures meant to diagnose thesolid-tumor cancer. Examples of such diagnostic procedures are variousimaging techniques or histological analysis of samples obtained duringbiopsy. Similar considerations apply to metastatic status and cancerstaging of the subjects with solid-tumor cancer. Metastatic status, suchas M1a or M1b status of NSCLC, and cancer staging can be determinedbefore, concurrently with or subsequently to the methods according tothe embodiments of the present invention, which are not limited by theorder of various diagnostic steps and procedures performed on thesubject.

The methods described herein can employ suitable diagnostic proceduresin addition to detection of mutated tumor-associated sequences in thesubject's blood, in order to accurately assess the status of thesolid-tumor cancer in the subject. Additional diagnostic procedures aresuitably selected to improve the accuracy of the assessment of thesolid-tumor cancer in the subject, and can include, but are not limitedto, various imaging techniques, biopsies, histological analysis,sequence analysis and other procedures.

The methods described herein are not limited to purely diagnosticprocedures, but can incorporate various treatment steps, thus embodyingapplication and use of the diagnostic discoveries described herein toimproved methods of treating solid tumor cancers, one example of whichis NSCLC. In one embodiment, the appropriate cancer treatments anddiagnostic procedures are suitably selected and administered orperformed based on the presence or absence of tumor-associated mutationsin the blood of a subject with a solid tumor cancer, such as thepresence or absence of tumor-associated EGFR mutations detected in theblood of NSCLC subject. Cancer treatments described herein can includesurgical or non-invasive treatments, such as drug or radiationtherapies.

Tumor-Associated Mutations

Tumor-associated mutations that are detected according to the methodsdescribed herein are mutations that are found in tumors of subjects withsolid tumor cancers and affect development of the solid tumor cancers inthe subjects. For example, tumor-associated mutations can affect theemergence, progression or recurrence of the cancer, as well as theresponsiveness or susceptibility of the cancer to a cancer therapy. Oneexample of tumor-associated mutations that can be detected according tothe methods described herein is the mutations in proto-oncogenes thatconvert them into oncogenes. Another example is the mutations intumor-suppressor gene that result in the loss or decrease of theirfunction. The mutations that can be detected according to the methods ofthe present invention are not limited to the mutations inprotein-encoding genes, but can also include the mutations in non-codingnucleic acid sequences, such as regulatory elements, sequences encodingnon-coding RNA, and other non-coding sequences. Tumor-associatedmutations of the protein-coding nucleic acid sequences can be in-framedeletions or insertions, as well as substitutions. For example, mutatedEGFR sequences being detected are typically nucleic acid sequences thatcontain one or more in-frame nucleotide deletions or insertions, as wellas nucleotide substitutions that result in mutated amino acid sequenceof EGFR. Tumor-associated mutations can result in protein fusions. Someexamples of tumor-associated mutations that can be detected in patient'sblood and used to monitor cancer emergence, progression, recurrence, aswell as to monitor cancer therapy, include without limitation, thefollowing mutations: EGFR mutations, KRAS mutations, including mutationsin KRAS codons 12, 13, 61 and 146, ALK mutations, including ALK fusions,ROS1, including ROS1 fusions, c-MET mutations, PIK3CA (PI3K-CA)mutations, NRF2 mutations, FGFR1-3 mutations, AKT1 mutations, includingAKT1 fusions, BRAF mutations, including V600E substitution, NRASmutations, TMPRSS2:ERG fusion, SPOP mutations, RET fusions, PPAR-gammafusions, IDH-1 mutations, and IDH-2 mutations. It is to be understoodthat some of the above mutations are associated with some, but notnecessarily all, of the solid-tumor cancers. Accordingly, detection ofsome of the above tumor-associated mutations can be more suitable forassessment of certain cancers. For example, detection of the followingmutations can be suitable for assessment of lung cancer: EGFR mutations,KRAS mutations, ALK fusions, ROS1 fusions, c-MET mutations, PIK3CA(PI3K-CA) mutations, NRF2 mutations and FGFR1-3 mutations. In anotherexample, detection of AKT1 mutations, including fusions, can be suitablefor assessment of breast cancer. In one more example, detection of KRASmutations, such as mutations of codons 12, 13, 61 and 146, BRAFsubstitution V600E, NRAS mutations. PIK3CA (PI3K-CA), EGFR extracellulardomain hot spot mutations can be used for assessment of colorectalcancer. Detection of TMPRSS2:ERG fusion and SPOP mutations can be usedfor assessment of prostate cancer. Detection of BRAF mutations, NRASmutations, RET fusion and PPAR gamma fusion can be used for assessmentof thyroid cancer. Detection of mutations in IDH-1 and IDH-2 can be usedfor assessment of glioblastoma, while detection of mutations in FGFR3can be used for detection of bladder cancer. It is to be understood thatthe above list of the associations of the tumor-associated mutations andtypes of cancers is not exhaustive or limiting.

Non-Small Cell Lung Cancer

Lung cancer is a solid tumor cancer that forms in lung tissue. Most ofthe lung cancer begins in the epithelial cells lining air passages. Thistype of cancer is termed “Non-Small Cell Lung Cancer” (NSCLC). Theother, less prevalent, type of lung cancer is termed “Small-Cell LungCancer,” which begins in the non-epithelial lung cells, such as nervecells or hormone-producing cells. The classification of the lung cancerinto NSCLC and small cell is important for determining an appropriatetreatment. Lung cancer is also described in terms of staging, whichdescribes the extent of cancer in a patient's body. In the currentclinical practice, lung cancer is typically staged according toClassification of Malignant Tumors (TNM), developed and maintained bythe International Union Against Cancer (UICC). TNM classification takesinto account the size of the tumor and whether it has invaded nearbytissue, involvement of regional lymph nodes, and distant metastasis, orspread of cancer from one body part to another. According to current TNMclassification of lung cancer is divided into five stages. Stage 0 isalso called in situ lung cancer, meaning that the cancer did not invadetissues outside the lung. Stage I lung cancer is a small tumor that hasnot spread to any lymph nodes and cam be completely surgically removed.Stage I is divided into two sub-stages. A and B, based on the size ofthe tumor. Small tumors, such as those less than 3 cm are classified asstage IA. Stage I tumors between 3 and 5 cm are typically classified asstage IB lung cancer. Stage II typically refers to larger tumors, withsub-stage IIA describing the tumors larger tumor (over 5 cm but lessthan 7 cm wide) that has spread to the lymph nodes or a larger tumor(more than 7 cm wide) that may or may not have invaded nearby structuresin the lung but has not spread to the lymph nodes.

When lung cancer metastasizes, it spreads through blood or lymph vesselsafter breaking away from a lung tumor. Stage III describes the cancertumors that are difficult to remove, because they spread to the tissuesoutside of the lung. Stage III cancers are classified as either stageIIA or IIIB. For many stage IIIA cancers and nearly all stage IIIBcancers, the tumor is difficult, and sometimes impossible, to remove.For example, stage IIIB lung cancer may spread to the lymph nodeslocated in the center of the chest, or invade nearby structures in thelung. Stage IV typically describes lung cancer that has spread to morethan one area in the other lung, the fluid surrounding the lung or theheart, or distant parts of the body by the process of metastasis. Theterms “stage IVA” can be used to describe lung cancer that spread withinthe chest, while the term “stage IVB” when it has spread outside of thechest. In general, surgery is not successful for most stage III or IVlung cancer. Lung cancer can also be impossible to remove if it hasspread to the lymph nodes above the collarbone, or if the cancer hasgrown into vital structures within the chest, such as the heart, largeblood vessels, or the main breathing tubes leading to the lungs. StageIII and IV lung cancer can be described as “late stage lung cancer” or“advanced lung cancer.”

Late stage or advanced lung cancer can be characterized in terms of itsmetastatic status or metastatic stage. For example, so-called metastasisstages M0 and M1 can be used to refer to the cancer's metastatic status.M0 metastatic status typically indicates that no metastasis of a lungtumor is detected in a patient. M1 status typically indicates thatmetastasis is detected. M1 metastatic status can be further subdividedinto stages M1a and M1b. Metastatic stage M1a is typically used todescribe metastatic lung cancer in which separate tumor nodule ornodules appear in a contralateral lung lobe, lung cancer tumors withpleural nodules or malignant pleural or pericardial effusions.Metastatic status of NSCLC cancer in a subject can be determined byvarious diagnostic procedures, including imaging techniques, such as PETscanning, or histological examinations of tissue samples obtained bybiopsy. Metastatic stage M1b is typically used to describe lung cancerwith distant metastasis in extrathoracic organs.

Epidermal Growth Factor Receptor

Epidermal Growth Factor Receptor (EGFR), which is also known as HER-1 orErb-B1, is an oncogene involved in development and progression of NSCLCin some patients. EGFR is a membrane-bound receptor protein of Erbfamily. EGFR comprises an extracellular ligand-binding domain, atransmembrane domain, and an intracellular domain that possessestyrosine kinase activity. EGFR is inactive in its monomeric state.Binding of a ligand leads to homo and heterodimerization of EGFR withother HER family members, followed by intermolecular tyrosinephosphorylation. Adaptor or signaling molecules bind to phosphorylatedEGFR, which triggers downstream intracellular signaling cascades.Examples of the signaling cascades triggered by EGFR are Akt, STAT andMAPK cascades. EGFR is known to promote growth of various cancers byseveral mechanisms, including, but not limited to, EGFR amplification,and mutational activation of EGFR.

Anti-cancer therapeutic drugs were developed that inhibit tyrosinekinase inhibitory activity of EGFR. Two of such drugs are smallmolecules gefitinib and erlotinib, which belong to the class ofquinazoline derivatives. Gefitinib and erlotinib were both shown toinhibit EGFR tyrosine phosphorylation. In the clinical studies that ledto approval of gefitinib and erlotinib, the drugs were shown to prolongsurvival in a relatively small subset of non-small cell lung cancer(NSCLC) patients after chemotherapy. Subsequent studies revealed thatmutations in EGFR tyrosine kinase domain were present in a portion ofNSCLC patients, and that these mutations were associated with clinicalresponsiveness to gefitinib and erlotinib. EGFR mutations which wereassociated with resistance to gefitinib and erlotinib were alsoidentified. Discussion of the early developments in the area of EGFRmutations in NSCLC patients and their connection to gefitinib anderlotinib therapies is found, for example, in Pao and Miller, Journal ofClinical Oncology. 23:2556-2568 (2005) and Rosell et al., Clin. Cancer.Res. 12:7222-7231, incorporated herein by reference. The presence orabsence of EGFR mutations in NSCLC patients can therefore serve as amarker suitable for assessing the status of NSCLC in patients, such asdetermining whether a particular patient's NSCLC is potentiallyresponsive to EGFR-directed therapy.

Known EGFR mutations associated with drug susceptibility or resistanceto known targeted drug therapies are generally located in thetyrosine-kinase domain of EGFR. Some of the known mutations areillustrated in FIG. 1, and in Table 1. Some of these mutations areclassified into “activating mutations,” which are known to enhance EGFRsignaling. Some of the activating EGFR mutations are associated withsensitivity to targeted drug therapies, such as tyrosine kinaseinhibitor therapies, and are sometimes referred to as “sensitizing”mutations. Examples of such mutations are in frame deletions EGFR exon19, and some amino-acid substitutions, such as L858R, L861Q andsubstitutions at G719, sometimes referred to as G719X, which include,but are not limited to G719A, G719C and G719S.

Other EGFR mutations are associated with resistance to tyrosine kinaseinhibitor therapies, and often arise in the course of the therapy. Suchmutations can be referred to as “resistance” mutations, examples ofwhich are in frame EGFR exon 20 insertions and T790M and S678I aminoacid substitutions. The methods described herein employ detection ofEGFR mutations, including activating and resistance mutations, in theblood of a subject with NSCLC.

EXAMPLES Example 1 Isolation of Nucleic Acids and PCR Amplification

All the samples were acquired from lung cancer (NSCLC) patients. Nucleicacid isolation was performed utilizing COBAS® DNA Sample Preparation Kit(Roche Molecular Diagnostics, Indianapolis, Ind.) according to themanufacturer's instructions. Real-time alleles-specific PCRamplification was performed on a COBAS® instrument using COBAS® EGFRMutation Test kit (Roche Molecular Diagnostics) according to themanufacturer's instructions. Briefly, the COBAS® kit contains threereaction mixtures, MMX1, MMX2, and MMX3, for allele-specific real-timePCR detecting various mutations in the human EGFR gene. MMX1 comprisesprimers and 6-carboxyfluorescein (FAM)-labeled probes for multipledeletions in exon 19 of the human EGFR gene (termed Ex19Del) andsubstitution mutation S7681 (JA270 signal). MMX2 comprises primers andprobes for substitution mutation L858R (FAM signal) and mutation T790M(JA270 signal). MMX3 comprises primers and probes for substitutionmutation L861Q (FAM signal), a set of substitution mutations G719X (HEXsignal) and multiple insertions in exon 20 of the human EGFR gene(Ex20Ins) (JA270 signal). Each reaction further comprises internalcontrol (IC) primers and probes targeting exon 28 of the human EGFR gene(Cy5.5 signal).

Example 2 Establishing a Calibration Curve for the Quantification of DNATargets

To calibrate the assay, varying amounts of genomic DNA were subjected toreal-time PCR amplification using the COBAS® EGFR Mutation Test kit.Twelve levels of genomic DNA were tested: 0.25, 0.5, 1, 2, 4, 8, 16, 32,64, 125, 250 and 500 ng/reaction. At each genomic DNA level, 120replicate PCR assays were run with internal control (IC) primers andprobes in three different multiplex PCR reaction mixtures included inthe kit (MMX1, 2 and 3, see Example 1). The resulting standard curve isshown in FIG. 2. On FIG. 2, the X-axis represents genomic DNA level andthe Y-axis represents cycle number corresponding to the cross point(C_(p)) achieved in the reaction. Based on the experimental dataillustrated by FIG. 2, the valid range of the internal control values(IC C_(p) range) was set at 20-32. In the selected valid C_(p) range,assay linearity was observed for all the reaction mixtures tested.

Example 3 Establishing a Cut-Off Limit for the Quantitative PCR Assay

For each reaction mixture, a measurable range within the valid IC C_(p)range was established using the data from non-specific amplification inthe absence of the true target occurring at later cycles of PCR, whichwas termed “breakthrough amplification.” For each reaction mixture,breakthrough amplification was observed with at least one set of primersand probes. For each target within each reaction mixture, the value ofC_(p)R was determined, which was the difference between the internalcontrol signal and the breakthrough signal, calculated as the differencebetween breakthrough C_(p) and internal control C_(p) observed in thesame reaction. For example, for Ex 19del target (illustrated in Table2), breakthrough occurred at the higher levels of genomic DNA tested,but C_(p)R was consistently high at these levels. The minimum C_(p)Robserved was selected as a cut-off value. The target Ex 19del signal wasconsidered positive (mutation detected) only if the IC value Cp was inthe valid range, as discussed in Example 2, and the C_(p)R value (thedifference between the target and the control signal) fell below thecut-off value of 17.7.

Alternatively, the cut-off may be set simply as the lowest breakthroughC_(p) observed in the calibration example. As illustrated in Table 2,for the S768I target, the target signal was considered positive(mutation detected) only if the IC value was in the valid range and thetarget C, value was below the breakthrough threshold of 34 cycles.

Example 4 Establishing a Calibration Curve for the Quantification of aMutant Target in the Presence of Wild-Type Genomic DNA Target

To approximate patients' samples containing cancer cells and normalcells, as well as genomic DNA, various amounts of each mutant targetdetectable by the assay (see Example 1) were combined with variousamounts of wild-type genomic DNA. Different amounts of the targetnucleic acid containing T790M mutation (2, 4, 8, 50, 100, or 200ng/reaction) were combined with different amounts of wild-type genomicDNA background (0.25, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125,250, and 500 ng/reaction). The target-specific C, obtained in theexperiment was then plotted against the amount of input target DNA. Thesignal for T790M-specific probe (JA270 C_(p)) obtained at differentlevels of target DNA was averaged and plotted against the log copynumber of the T790M mutant target present in the sample. The resultingcalibration curve is shown in FIG. 3.

Example 5 Detecting Mutant EGFR DNA in the Blood of Lung Cancer (NSCLC)Patients

Blood plasma samples were collected from NSCLC patients after theyunderwent chemotherapy and before and during erlotinib targeted therapy.The timeline of the sample collection is schematically illustrated inFIG. 4. The samples were collected every four weeks at the time pointsindicated as CP₀₋₄ in FIG. 4. Sample collection did not necessarily stopat time point CP₄. DNA was isolated from the collected blood plasmasamples and subjected to real-time PCR amplification using the COBAS®kits in accordance with manufacturer's instructions (see Example 1). Forillustrative purposes, FIG. 5 schematically shows measured levels of anactivating exon 10 deletion (Ex19Del) and T790M activating substitutionof EGFR in blood plasma of two exemplary patients (“Case A” and “CaseB”). In both patients, tissue tumor samples obtained at the initialdiagnosis had been previously determined to contain an activating EGFRmutation (Ex19del) but no resistance mutation (T790M). The amount ofmutant DNA sequences, expressed in number of copies and plotted onY-axis of the plot shown in FIG. 5, was measured using the calibrationcurves described in Example 5. In both cases A and B, increase in theamount of mutant DNA in the blood correlated with progression of NSCLCas detected by suitable imaging techniques and also indicated the riseof resistance to erlotinib therapy.

Example 6 Detection of EGFR Mutations in the Blood of NSCLC Patientswith Different Metastasis Statuses

Two studies were conducted that correlated detection of EGFR mutationsin the blood plasma of NSCLC patients with the patient's metastasisstatus. In the first study (Study I), plasma samples and matching tissuesamples were collected from twenty eight Stage IV NSCLC patients.Mutation status of the tissue and blood samples was determined. The dataon the mutations detected in was compared to the metastasis status ofthe patients. Study I experimental data is summarized in Tables 3 and 4.

In the second study (Study II), plasma samples and matching tissuesamples were collected from seventeen Stage IV NSCLC patients. Mutationstatus of the tissue and blood samples was determined. The data on themutations detected in was compared to the metastasis status of thepatients. Study II experimental data is summarized in Tables 5-II, 5-IIand 6.

In both Study I and Study II, it was observed that positive agreementbetween detection of EGFR mutations in tissue and plasma samples wassignificantly higher for the patients with distant metastasis(metastatic status pM1b) than for the patient without distant metastasis(metastatic status pM1a). Summary of Study I and Study II data ondetection of activating EGFR mutations in blood of NSCLC patients ofdifferent metastatic status is schematically shown in Table 7.

Example 7 Benefits of Detecting EGFR Mutations in the Blood of InitiallyDiagnosed NSCLC Patients

EGFR activating mutations are detected in blood of 200 patients withinitially diagnosed stage IIIB through stage IV NSCLC. The detection isgenerally performed according to the procedures described in the earlierexamples. Activating EGFR mutations are detected in the blood of 20% ofthe patients. Resistance EGFR mutations are detected in a subset of thepatients carrying activating EGFR mutations. Metastasis status of thesepatients is determined by PET Scan. 50% of the patients are determinedto have pM1a metastasis status, and 50% are determined to have pM1bmetastasis status. Based on the knowledge of the high positive agreementbetween detection of the activating EGFR mutations in blood and theirpresence in tumor tissue in pM1b patients but not in pM1a patient,targeted tyrosine kinase inhibitor (TKI) therapy is recommended for andadministered to pM1b and pM1a patients with detectable EGFR activatingmutations in blood without additional diagnostic procedures. TargetedTKI therapy is not recommended for pM1b patients without detectable EGFRactivating mutations or with detectable resistance mutations in blood(no additional diagnostic procedures are deemed necessary). pM1apatients without detectable EGFR activating mutations in blood aredirected to biopsy of the tumor tissue with subsequent mutationdetection in the biopsy samples in order to determine whether or notthese patients are candidates for EGFR therapy. The abovedecision-making process for 200 patients is schematically illustrated inFIG. 6. Under this decision-making process, only 94 patients out of 200need biopsy followed by tissue mutation testing in order to determinewhether or not they are candidates for TKI targeted therapy.

Example 8 Benefits of Detecting EGFR Mutations in the Blood of RelapsedNSCLC Patients

EGFR activating mutations are detected in blood of 200 relapse patientswith stage IIIB through stage IV NSCLC. The detection is generallyperformed according to the procedures described in the earlier examples.Activating EGFR mutations are detected in the blood of 20% of thepatients. Metastasis status of these patients is determined by PET Scan.40% of the patients are determined to have pM1a metastasis status, and60% are determined to have pM1b metastasis status. Based on theknowledge of the high positive agreement between detection of theactivating EGFR mutations in blood and their presence in tumor tissue inpM1b patients but not in pM1a patient, targeted tyrosine kinaseinhibitor (TKI) therapy is recommended for and administered to pM1b andpM1a patients with detectable EGFR activating mutations in blood withoutadditional diagnostic procedures. Targeted TKI therapy is notrecommended pM1b patients without detectable EGFR activating mutationsin blood (no additional diagnostic procedures are deemed necessary).pM1a patients without detectable EGFR activating mutations in blood aredirected to biopsy of the tumor tissue with subsequent mutationdetection in the biopsy samples in order to determine whether or notthese patients are candidates for EGFR therapy. The abovedecision-making process for 200 patients is schematically illustrated inFIG. 7. Under this decision-making process, only 74 patients out of 200need biopsy followed by tissue mutation testing in order to determinewhether or not they are candidates for TKI targeted therapy.

Example 9 Benefits of Detecting EGFR Mutations in the Blood of NSCLCPatients

EGFR activating mutations are detected in blood of 200 relapse patientswith stage IIIB through stage IV NSCLC. The detection is generallyperformed according to the procedures described in the earlier examples.The decision-making process is schematically illustrated in FIG. 8.

TABLE 1 Examples of EGFR mutations. Mutation Amino Acid Change Exon 2155G>A G719S 18 2155 G>T G719C 18 2156 G>C G719A 18 2233_2247del15K745_E749del 19 2235_2248>AATTC E746_A750>IP 19 2235_2249del15E746_A750del 19 2235_2251>AATTC E746_T751>IP 19 2235_2252>AATE746_T751>I 19 2235_2255>AAT E746_S752>I 19 2236_2250del15 E746_A750del19 2236_2253del18 E746_T751del 19 2237_2251del15 E746_T751>A 192237_2252>T E746_T751>V 19 2237_2253>TTGCT E746_T751>VA 192237_2254del18 E746_S752>A 19 2237_2255>T E746_S752>V 19 2237_2257>TCTE746_P753>VS 19 2238_2248>GC L747_A750>P 19 2238_2252del15 L747_T751del19 2238_2252>GCA L747_T751>Q 19 2238_2255del18 E746_S752>D 192239_2247del9 L747_E749del 19 2239_2248>C L747_A750>P 19 2239_2251>CL747_T751>P 19 2239_2253del15 L747_T751del 19 2239_2256del18L747_S752del 19 2239_2256>CAA L747_S752>Q 19 2239_2258>CA L747_P753>Q 192240_2251del12 L747_T751>S 19 2240_2254del15 L747_T751del 192240_2257del18 L747_P753>S 19 2253_2276del24 S752_I759del 19 2303 G>TS768I 20 2307_2308 ins 9(GCCAGCGTG) V769_D770insASV 202309_2310(AC<CCAGCGTGGAT V769_D770insASV 20 SEQ ID NO: 1) 2310_2311 insGGT D770_N771insG 20 2311_2312 ins 9(GCGTGGACA) D770_N771insSVD 202319_2320 ins CAC H773_V774insH 20 2369 C>T T790M 20 2573 T>G L858R 212573-2574TG>GT L858R 21

TABLE 2 Summary of the exemplary experimental data used for establishingcut-off limits for a measurable range of quantitative PCR assays.Breakthrough Data Ex 19 Deletion S768I # of # of Genomic reactionsAverage Minimum reactions Average Minimum DNA, with break- break- withbreak- break- ng per # of Average observed through through AverageMinimum observed through through Average Minimum reaction reactions ICC_(p,)

C_(p) C_(p) C_(p)R C_(p)R

Cp Cp C_(p)R C_(p)R 500 120 19.89 1 40   40   20.19 20.19 4 39.01 38  19.06 17.82 250 120 20.81 3 40.42 38.79 19.65 18.16 0 NA NA NA NA 125120 21.72 0  NA* NA NA NA 1 34.01 34.01 12.27 12.27 62.5 120 22.67 140.37 40.37 17.7  17.7  1 39.02 39.02 16.18 16.18 31.3 120 23.65 0 NA NANA NA 2 34.53 34.19 10.84 10.56 15.6 120 24.67 0 NA NA NA NA 0 NA NA NANA 7.8 119 25.74 0 NA NA NA NA 2 34.36 34.29  8.44  8.41 3.9 119 26.87 0NA NA NA NA 0 NA NA NA NA 2 119 28.05 0 NA NA NA NA 0 NA NA NA NA 1 12029.28 0 NA NA NA NA 0 NA NA NA NA 0.5 107 30.50 0 NA NA NA NA 0 NA NA NANA 0.25 107 31.61 0 NA NA NA NA 0 NA NA NA NA *NA stands for “notapplicable”

indicates data missing or illegible when filed

TABLE 3 Summary of Study I experimental data. EGFR Activating MutationsPlasma MUT+* Plasma MUT−** Tissue 13 (metastasis status of 2 3 MUT+samples not known) pM1a pM1b*** pM1a pM1b 2 9 3 0 *MUT+ = activatingmutation detected **MUT− = activating mutation not detected***Metastasis status of some of the patients was not known

TABLE 4 Summary of Study I experimental data for the patients withdetectable mutations in tissue samples. Mutations Mutations detected indetected in Metastasis Patient ID tissue sample plasma sample status 1L858R & T790M L858R & T790M N/D* 2 Ex19Del Ex19Del N/D 3 L858R L858RpM1b 4 S768I, G719X S768I & G719X pM1b 5 L858R L858R pM1b 6 Ex19DelEx19Del pM1a 7 Ex19Del Ex19Del & pM1b T790M 8 Ex19Del Ex19Del pM1b 9Ex19Del Ex19Del pM1b 10 Ex19Del Ex19Del pM1b 11 Ex19Del Ex19Del pM1b 12Ex19Del Ex19Del pM1b 13 L858R L858R pM1a 14 L858R & Ex20Ins — pM1a 15L858R and T790M — pM1a 16 Ex19Del & T70M — pM1a *N/D = not determined

TABLE 5-I Summary of Study II experimental data EGFR ActivatingMutations Plasma MUT+* Plasma MUT−** Tissue MUT+ 11 4 pM1a*** pM1b pM1apM1b 1 9 3 1 *MUT+ = activating mutation detected **MUT− = activatingmutation not detected ***Metastasis status of some of the patients wasnot known

TABLE 5-II Summary of Study II experimental data EGFR resistancemutation T790M Plasma MUT+* Plasma MUT−** Tissue MUT+ 6 3 pM1a pM1b pM1apM1b 1 5 2 1 *MUT+ = activating mutation detected **MUT− = activatingmutation not detected ***Metastasis status of some of the patients wasnot known

TABLE 6 Summary of Study II experimental data Mutations detected inMutations detected in Patient ID tissue sample plasma sample Metastasisstatus 1 Ex19Del Ex19Del pM1b 2 L861Q & G719X & L861Q & G719X & pM1bT790M T790M pM1b 3 Ex19Del Ex19Del pM1b* 4 L858R L858R pM1b 5 L858R &T790M L858R & T790M pM1b 6 L858R & T790M L858R & T790M pM1b 7 Ex19Del &T790M Ex19Del & T790M pM1b 8 Ex19Del & T790M Ex19Del & T790M pM1b 9L858R L858R pM1b 10 Ex19Del & T790M Ex19Del & T790M pM1a 11 Ex19Del &T790M Ex19Del & T790M N/D 12 T790M (Ex19Del) — pM1a 13 Ex19Del & T790M —pM1a 14 Ex19Del & T790M — pM1b 15 Ex19De — pM1b 16 — Ex19Del pM1b 17 —Ex19Del pM1b *N/D = not determined

TABLE 7 Summary of Study I and Study II data on detection of activatingEGFR mutations in blood of NSCLC patients of different metastasisstatus. Metastasis Positive Negative Overall Study status agreementagreement agreement I Overall*  81% 100% 89% pM1a  40% pM1b 100% IIOverall*  73%  0% 65% pM1a  25% pM1b  90% *Includes patients with pM1a,pM1b and non-determined metastasis status

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of identifying a candidate non-small cancer cell lung cancer(NSCLC) patient for a targeted drug therapy, comprising: detectingpresence or absence of one or more mutated Epidermal Growth FactorReceptor (EGFR) sequence in blood from the patient; assessing metastaticstatus of the NSCLC patient as M1a or M1b; and, identifying the patientas a candidate for the targeted drug therapy based on at least thedetected presence of the one or more mutated EGFR sequence in the bloodof the patient, and the metastatic status of NSCLC in the patient. 2.The method of claim 1, wherein the one or more mutated EGFR sequencecomprises an activating EGFR mutation.
 3. The method of claim 2, whereinthe activating EGFR mutation is selected from the group consisting of anexon 19 deletion, L858R, L861Q and G719X
 4. The method of claim 1,wherein the one or more mutated EGFR sequence comprises a resistanceEGFR mutation.
 5. The method of claim 4, wherein the resistance EGFRmutation is selected from the group consisting of T790M, S678I and anexon 20 insertion.
 6. The method of claim 1, wherein the detectingcomprises testing the blood from the patient for the one or more mutatedEGFR sequence by an analytical technique.
 7. The method of claim 1,wherein the targeted drug therapy is a tyrosine kinase inhibitor.
 8. Themethod of claim 7, wherein the tyrosine kinase inhibitor is erlotinib ofgefitinib.
 9. The method of claim 1, wherein the assessed metastaticstatus of the patient as M1a.
 10. The method of claim 9, wherein theidentifying further comprises detecting presence or absence of one ormore mutated EGFR sequence in a tumor tissue of the patient with themetastatic status M1a if the absence of the at least one of the one ormore mutated EGFR sequence in the blood of the patient is detected. 11.The method of claim 1, wherein the assessed metastatic status of theNSCLC patient is M1b.
 12. The method of claim 11, wherein theidentifying comprises identifying the NSCLC patient with the metastaticstatus M1b as the candidate for the targeted drug therapy if thepresence of the at least one of the one or more mutated EGFR sequence inthe blood of the patient is detected.
 13. The method of claim 12,wherein the assessing comprises administering a targeted drug therapy tothe subject if the presence of the at least one of the one or moremutated EGFR nucleic acid sequence in the blood of the subject isdetected.
 14. The method of claim 1, wherein the assessing comprisesadministering a targeted drug therapy to the subject if the presence ofthe at least one of the one or more mutated EGFR nucleic acid sequencein the blood of the subject is detected.
 15. The method of claim 14,wherein the at least one or more mutated EGFR sequence contains anactivating EGFR mutation and the targeted drug therapy is a tyrosinekinase inhibitor.
 16. The method of claim 1, wherein the detectingcomprises detecting one or more times the presence or absence of one ormore mutated EGFR sequence in the blood of the subject before, during,or after targeted drug therapy, or any combination thereof.
 17. Themethod of claim 16, wherein the presence of at least one or more mutatedEGFR sequence containing an activating EGFR mutation is detected and theassessing further comprises increasing a dose of a tyrosine kinaseinhibitor administered to the patient if an increase in quantity of themutated EGFR sequence is detected.
 18. The method of claim 1, whereinthe assessing comprises modifying the targeted drug therapy if presenceof a resistance EGFR mutation is detected.
 19. The method of claim 1,wherein the detecting comprises detecting the presence or the absence ofthe one or more mutated EGFR sequence in the blood of the NSCLC patientbefore, during, or after chemotherapy, before or after surgery or anycombination thereof.
 20. A method of assessing status of a subject witha solid tumor cancer, comprising: detecting presence or absence of oneor more tumor-associated mutated nucleic acid sequence in blood from thesubject with the solid tumor cancer; and, assessing the status of thesubject with distant metastasis solid tumor cancer based on the detectedpresence or absence of the one or more mutated tumor-associated nucleicacid sequence.
 21. The method of claim 20, wherein the assessingcomprises administering a targeted drug therapy to the subject if thepresence of the at least one of the one or more mutated tumor-associatednucleic acid sequence in the blood of the subject is detected.
 22. Themethod of claim 21, wherein the at least one or more mutated oncogenesequence contains an activating mutation and the targeted drug therapyis a tyrosine kinase inhibitor.